Concerning the Characters of Certain Fungi 
as Exhibited by their Growth in the 
Presence of other Fungi 


BY 


Ghiovigiotiou LYMAN. PORTER 


B.S. Illinois Wesleyan, I91I 
A.B. University of Illinois, 1913 
A.M. University of Illinois, 1921 


THESIS 


SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS 
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BOTANY 
IN THE GRADUATE SCHOOL OF THE 
UNIVERSITY OF ILLINOIS 
1923 


Reprinted from 
AMERICAN JOURNAL OF BOTANY 
Vol. XI, No. 3, Pages 168-188 


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Reprinted from the AMERICAN JOURNAL OF BoTANy, XI: 168-188, March, 1924. 


CONCERNING THE CHARACTERS OF CERTAIN FUNGI 
AS EXHIBITED BY THEIR GROWTH IN THE 
PRESENCE, OF -OFHER FUNGI 


CHARLES LYMAN PORTER 


(Received for publication June 23, 1923) 


INTRODUCTION 


In common with all other organisms, fungi are modified by the changes 
in their environment. The presence of other living forms constitutes a 
part of the environment, and consequently these affect the morphology 
and physiology of the fungi; thus, growth is usually checked when two or 
more fungi are contiguous. Such inhibition may be mutual, or the growth 
of one individual may be inhibited more than that of the other. The antag- 
onistic action .of one fungus toward another may be due to a variety of 
causes, and results in numerous modifications of the organisms involved. 
The study of growth changes so induced is the purpose of this paper. 


METHODS 


In the routine work of determining types of inhibition, the various 
organisms used were grown on cornmeal agar in petri plates. Effort was 
made always to have the conditions as nearly uniform as possible. Varia- 
tions were made from this routine procedure in order to observe the growth 
phenomena under different conditions. 

A. The depth of the medium was varied by permitting the agar to 
harden in the plates while the plates were tilted. Colonies were then planted 
in the shallow and deep regions of the agar. 

B. The medium was varied as to the amount and kinds of nutrients 
present, emploving washed agar, plain agar, cornmeal agar, dextrose agar, 
and Brazil-nut agar. 

C. The time element was varied by making inoculations at periods 
ranging from 24 to 129 hours from the time of the initial transfer. 

D. The amount of inoculum was varied. | | 

In noting morphological changes, direct observations were made through 
the microscope. One other method that proved very successful was as’ 
follows: Thin layers of agar were carefully poured on cooled sterile cover 
slips, where the agar hardened immediately. Slips thus prepared were 
inoculated with two fungi that were to be placed under observation. The 
inoculations were made so that the developing colonies would not be more 
than I to 2 cm. from each other. As soon as development was well started, 
the cover slips were mounted on the stage of the microscope and the mi- 

168 


169 AMERICAN JOURNAL OF BOTANY [Vol. 11, 


croscope was placed in position on the stand of a Bausch and Lomb photo- 
micro-projection apparatus. In this manner the fungous hyphae were 
enormously magnified, and even minute changes in appearance and the 
manner and quality of the change could be quickly noted. Observations 
were made at short intervals, and the light was not kept on longer than 
necessary to make observations lest the illumination modify the growth in 
some manner. 

Aseptic seedlings were secured and placed in rag-dolls in the manner 
suggested by F. L. Stevens (41). The dolls were divided into four lots, 
the lots being subjected to the following treatments: (1) Several loopfuls 
of Helminthosporium spores were rubbed on the outside of the cloth sur- 
rounding the seedlings, and the whole roll was then dipped into a broth 
suspension of organism no. 45. (2) Treated as lot one, except that the rolls 
were not dipped into the broth suspension. (3) Rolls containing the seed- 
lings were dipped into the broth suspension but were not inoculated with 
the spores of Helminthosporium. (4) Rolls were placed in their tubes with- 
out being inoculated with the spores of Helminthosporium or dipped into 
a broth suspension of organism no. 45. Lots 3 and 4 served'as controls. 

Pot cultures were secured as follows: Five-inch pots were filled with 
earth. Cones, one inch in diameter at the top and three and one half inches 
long, made of thin paper, were filled with earth which had been thoroughly 
moistened with a broth culture of organism no. 45. These were sunk in the 
center of the pots. Inside these cones were placed a number of flax seeds 
that had been so treated as to render them aseptic. The soil outside the 
cones was inoculated with the spores of Fusarium lint. In the control pots 
the soil in the cones was moistened with sterile broth only. 

The staling products of Penicillium growth were secufed in the follow- 
ing manner: Sterile orange juice was inoculated with Pentcillium italicum 
and kept at room temperature for six weeks. At the end of this time the 
juice was filtered through a Reichel filter. The filtrate was placed in sterile 
petri dishes and permitted to stand for several weeks in a sterile desiccator 
over calcium chlorid. In this time the filtrate was considerably concentrated. 
The liquid was injected into the oranges with a Leur syringe. 


ACCESSION LIST OF ORGANISMS 


The following organisms are those made use of in my experiments. 
Their history so far as it is known may be found in a typewritten thesis 
filed in the library of the University of Illinois. The organisms are numbered 
throughout the text to correspond with the numbers of the accession list. 


NHAUPW DN 


Pink Fusarium 
Rhizopus nigricans 


. Actinomyces 


. Sclerotium rolfsit 8. Pink yeast 
. Actinomyces g. White yeast 
. Helminthosporium 10. White yeast 


. Penicillium glaucum 
. Bacterium 
. Penicillium italicum 


Mar., 1924] 


PORTER — CHARACTERS OF CERTAIN FUNGI 170 


14. Penicillium digitatum 90. Bacillus prodigiosus 

25. Helminthosporium gl. Bacterium alcaligenes 

26. Helminthosporium 92. Bacillus capsulatus 

29. Alternaria 93. Actinomyces albus 

30. Mucor 94. Bacillus ramosus 

31. Bacteria 97. Bacillus vulgatus 

32. Bacteria 98. Bacterium megatherium 
33. Bacteria 100. Cladothrix dichotoma 

34. Gliocladium 103. Fusarium 

35. Colletotrichum lindemuthianum 104. Helminthosporium sativum 
36. Helminthosporium 105. Fusarium lint 

37. Helminthosporium 106. Pilobolus-like fungus 

39. Sterigmatocystis 108. Alternaria 

40. Alternaria 110. Zygorhynchus 

42. Penicillium 111. Syncephalastrum 

44. Actinomyces 112. Cunninghamella 

45. Bacterium! 113. Rhizopus nigricans, plus race 
46. Acrothecium 114. Rhizopus nigricans, minus race 
50. Unknown organism 115. Actinomyces tricolor 
*.53. Lactobacillus 116. Actinomyces albus var. ochraceus 
58. Azotobacter 117. Actinomyces nigrificans 
61. Acrostalagmus cinnabarinus 118. Phyllosticta solitaria 

62. Cytosporium ribis 120. Fusarium culmorum 

68. Fusarium coeruleum 121. Gloeosporium piperatum 
70. Sporotrichium bombycinum 122. Colletotrichum nigrum 

‘71. Sclerotinia libertiana 123. Ustilago violacea 

72. Botrytis 124. Ustilago violacea 

73. Fusarium lint 125. Alternaria crassa 

74. Bacillus carotovorus 127. Bacillus mesentericus 

77. Fusarium lint 128. Helm:nthosporium 

86. Bacillus proteus 129. Fusarium lint 

87. Sarcina lutea 130. Fusarium lint 

88. Sarcina aurantiica 131. Fusarium lint 

89. Pseudomonas violaceus 132. Pythium 


TYPES OF INHIBITION 


There are many conceivable reactions when the mycelium of one fungus 
approaches that of another. These theoretical possibilities are nearly all 
demonstrated by actual examples. Moreover, these examples occur fre- 
quently enough and, under properly controlled experiments, regularly 
enough, to suggest that they may be classified into types which occur when- 
ever the same organisms are made to react with each other. For conven- 
ience in discussion, I have described the most prominent and common types 
of reaction and have designated them by letters. 

Type A (text fig. 1, A). Mutually intermingling. It might be assumed 
that this would be a common type, but few really good examples are found. 
Perhaps the best is afforded by the intermingling hyphae of two colonies of 
Rhizopus. Organism no. 5 grown against no. 5 also presents a fairly good 
example of this type. It seems, however, but rarely possible for two organ- 
isms to occupy equally well the same area at the same time. 


1 The index number of this organism was determined and reads 5131-52120-1333. 


T71 AMERICAN JOURNAL OF BOTANY [Vol. "ra; 


Type B (text fig. 1, B). Growth superficial over the contending or- 
ganism. The underlying organism is always greatly inhibited. An ex- 
ample is supplied by organism no. 2 grown with no. 4. No. 4 is the inhibited 
organism. 


; & 45 


Text Fic. 1. Types of inhibition. A,mutuallyintermingling. B,'overgrowing. C, 
slight inhibition. D, growth around. 4, inhibition at a distance. 


Type C (text fig. 1, C). Slight inhibition. Both organisms are inhibited 
but approach each other until almost in contact, when growth ceases. In 
such cases the space between the two colonies, while very narrow, is clearly 
marked. This is the prevailing type when any organism is grown with 
another individual of the same species. 

Type D (text fig. 1, D). Growth around the contending organism. The 
Helminthosporiums react in this manner when grown with organisms 
numbers 8, 9, or IO. | 

Type E (text fig. 1, #). Mutual inhibition at considerable distance. 
Organisms nos. 31, 33, and 34 so inhibit any of the Helminthosporiums. 
These three organisms when grown with organisms nos. 5, 30, 32, and 35 
produce this type of inhibition. The power to inhibit strongly which is 


Mar., 1924] PORTER — CHARACTERS OF CERTAIN FUNGI 172 


shown by nos. 31, 33, 34, and 45 will be a fruitful field of study for future 
investigations. They can inhibit growth in many fungi at a distance of 100 
mm., and perhaps at greater distances. 

Very often a fungus may show for a time complete checking of growth 
when in the presence of another fungus. Later, growth will appear to be 
resumed. This growth on closer examination will be seen to be in the deeper 
part of the medium, which is apparently less affected by the inhibiting 
organism. The surface growth when once checked usually does not again 
resume activity. A case of this sort is illustrated by text figure 2. 


TExT Fic. 2. A, colony of Alternaria. B, bacterial growth. XY represents the 
line upon which the Alternaria colony was temporarily inhibited. 


COLONY CHANGES 


Inhibition modifies a fungus in many ways evident to the eye. Phys- 
iological changes also probably occur, but these will not be discussed at this 
time. Changes that occur vary directly with the inhibition. All organisms 
that do not overspread the surface of the contesting fungus are restricted 
in growth. Helminthosporium when alone in a plate would normally con- 
tinue to grow until the surface of the medium is occupied. If any other 
organism is placed in the plate with the Helminthosporium, the growth of 
the latter is greatly restricted. Two spores of Helminthosporium may 
germinate in the same plate and the resulting colonies may each cover 
approximately half the area, with a narrow but definite streak of unoccupied 
agar between them. If organism no. 31, no. 33, or no. 34 is planted ina 
petri plate with Helminthosporium, the latter forms a colony with a curved 
margin, the concave side toward the antagonistic organism. 

Some fungi are almost entirely inhibited in growth by the presence of 
other organisms in the same petri plate. This reaction is illustrated by 
organisms nos. 3, 7, 8, 9, or 10 in the presence of Helminthosporium, or of 
any other fungus that develops mycelium rapidly. Thus we see that an- 
tagonism greatly modifies the shape and size of the colony. 


173 AMERICAN JOURNAL OF BOTANY [Vol. rf 


Changes in color are quite often associated with other inhibitional 
changes. Color may be limited to a very narrow band or may pervade the 
entire mycelium. 

Organism no. 12, a bacterial form, causes a narrow (I-mm.) line along 
the proximal border of an Helminthosporium colony with which it is grown. 
Organism no. 5 causes a similar red line to appear in a colony of organism 
no. 4 when these two are grown together in a petri plate. Organism no. 5, 
when grown by itself, has a light pinkish tinge. The depth of this color is 
modified by the organism grown with it; it may fade out entirely or become 
bright red, with all gradations between according to the species of the an- 
tagonistic organism. 

Antagonism stimulates spore-production. Along the line of impending 
contact between colonies spores are more numerous than elsewhere. No 
case was observed in which spore-production was decreased, although such 
spore-production is more apparent with some combinations than with 
others. | 

MORPHOLOGICAL CHANGES 


When Helminthosporium is growing in pure culture on cornmeal agar 
under normal conditions of moisture and temperature, the aérial mycelium 
at the edge of the colony is of uniform diameter, infrequently branched, 
and straight. If a culture of organism no. 45 is introduced several centi- 
meters away from the periphery of the colony, the following morphological 
changes occur: when the hyphae of Helminthosporium are within 2 centi- 
meters of the bacterial colony, growth slackens and eventually ceases. 
Branches are given off from the hyphae in every direction and these become 
gnarled and twisted, and bubble-like enlargements of varying size appear 
in this portion of the mycelium with almost instantaneous suddenness. The 
swollen segments may be regarded in some cases as extremely dwarfed 
branches, but often they appear as enlargements in the existing hyphae. 
So great is the distortion thus produced that often the species is not recogniz- 
able. The multiplication of branches piles up the hyphae along the line of 
impending contact, which appears to the eye as a black streak. The effect 
is often heightened by excessive spore-formation along thisline. In addition 
to these distortions, the hyphae have a tendency in their general direction 
of growth to avoid the zone of influence controlled by organism no. 45 and 
to grow around it. Eliiot (12), working with Alternaria and a bacterial 
form which he designated as ‘‘X,’’ found distortions practically identical 
with those which I have described for Helminthosporium, and I have 
obtained similar results with Alternaria when inhibited by my organism 
no. 45. No. 45 produces strong inhibitory effects under the conditions 
described in all filamentous fungi experimented with except the Phycomy- 
_cetes, which are little affected. 

The greater the inhibition, the greater the distortion. Therefore, the 
greater the distance through which the reaction takes place, the more marked 


Mar., 1924] PORTER — CHARACTERS OF CERTAIN FUNGI 174 


are the results. The morphological changes grade from changes in form, 
size, and structure of the hyphae, and change in direction of growth, to mere 
cessation of growth with hyphal segments becoming progressively shorter. 

The morphological changes described above indicate that there are 
forces operating capable of producing profound effects on the morphology 
of the organism involved. In every case in which malformations occur, no 
morphological differences of diagnostic value could be determined. Three 
hypotheses suggest themselves as to the causes of these variations: (a) The 
nutrients may be exhausted. (b) The distortions may be due to change in 
the osmotic equilibrium of the medium induced by the metabolic activities 
of the growth process. (c) Certain poisonous products may be created 
by fungous growth capable of producing malformations and creating a 
zone through which fungous filaments can not pass. Much work would have 
to be done in order to determine which of these hypotheses best explains 
the facts. I am not prepared to give a definite answer to the question, but 
my experiments have thrown some light that may be of aid in the solution. 

It has been explained that organism no. 45 inhibits all Helminthospor- 
iums at a distance of 2 centimeters or more, so that the growth of the latter 
is checked entirely. If a block of the medium which occupies the space 
between the two organisms is removed, aseptic precautions being exercised, 
and placed in another dish occupied solely by an Helminthosporium colony, 
the filaments of the Helminthosporium will not pass over the block but will 
be checked sharply in front of it, with characteristic distortions. That the 
effect was not due to mechanical blockade was proven when sterile blocks 
of normal agar were placed in proximity to Helminthosporium colonies and 
were soon overgrown by the advancing hyphae. Furthermore, blocks of 
agar taken from the vicinity of organism no. 45 caused inhibition with 
effects as marked as before when the blocks were inlaid rather than placed 
upon the surface (text fig. 3). 


zt 
a 


A 


TeExT Fic. 3. _ Colony of Helminthosporium sharply inhibited in front, of B, a block of 
sterile agar taken from the vicinity of organism no. 45. Blocks A and C are from sterile 
agar plates and cause no inhibition. . 


i175 AMERICAN JOURNAL OF BOTANY [Vol. 11 


To note the effects of certain chemicals on the growth of Helmintho- 
sporium filaments, the following substances were used: 
1. Copper sulphate crystals, 

2. Phenol crystals, d 

3. Phenol (melted) full strength, 

4. Phenol (melted) one-half strength, 

5. Chloramine T crystals, 

6. Sodium nitrate, 

7. Mercuric chlorid, 

8. Glucose, 

9. 95 percent alcohol (one drop), 

IO, II, 12, 13, 14. Aqueous solutions of copper sulphate of the following strengths: satu- 
rated, 75 percent, 50 percent, 25 percent, and I percent. 


These materials were placed within 2 centimeters of vigorously growing 
colonies of Helminthosporium no. 20. The chemicals were permitted to 
act separately in separate plates. Total inhibition was obtained by the 
use of substances I, 2, 3, 4, 5, 7, 10, 11, 12, and 13. With substances 6 and 
14, slight inhibition was obtained. Substances 8 and 9 caused no inhibition. 
Where inhibition occurred, except with 6 and 14, distortion of the mycelium 
was apparent. The distortion was in direct ratio to the strength of the 
substances used. The distortions were very similar to those previously 
described when organism no. 45 was used as the inhibiting agent. Those 
occurring with the use of chemicals very often showed hyphae peculiarly 
twisted into tight loops. It will be noted that in this experiment those 
chemicals which are known as powerful germicides and fungicides produced 
the more marked effects, even though considerably diluted. 


EFFECT OF MODIFICATIONS UPON INHIBITION 


To this point, the phenomena peculiar to antagonism have been con- 
sidered as occurring under uniform conditions. In order to discover whether 
variation of conditions materially changes the nature and degree of inhibi- 
tion, modifications in technique were introduced. 

The depth of the medium in a plate was easily varied through all grada- 
tions by the simple expedient of pouring the medium in the plates while 
they were tilted. A colony of a fungus planted in such a plate tended to 
occupy all sections, and the effect of the presence of other fungi could be 
tested in both the shallow and the deep portions. Media including cornmeal 
agar, dextrose agar, plain agar, and washed agar were experimented with 
in this manner. In all cases in which inhibition occurred normally it was 
more marked in the shallow portion of the plate than in the deep portions. 
Evidently the normal inhibitional effect was heightened by the lack of 
nutrients in the shallow situations. Furthermore, in a normally poured 
plate the effect of surface inhibition is often overcome by the inhibited 
fungus sending forward hyphae in the deeper portions of the medium where 
the inhibitory products make their presence felt to a less degree. In a 


Mar., 1924] PORTER — CHARACTERS OF CERTAIN FUNGI 176 


shallow medium, however, the inhibitory products penetrate throughout the 
layer of the medium and the fungus is checked entirely. While the effects 
are more marked in the shallow portions, they are always the same in char- 
acter and the inhibitional type is not changed. Spore-formation was more 
abundant in the shallow portions, and especially in the direction of approach- 
ing contact, than was normally the case. ; 

Two types of media were used: one rich in nutrients, the other poor in 
nutrients. Cornmeal, dextrose, and brazil-nut agars represent the former 
type, while plain and washed agars are included in the latter class. Many 
organisms were grown on these media in a manner to test their inhibitional 
characteristics. The organisms included Helminthosporium, bacteria, 
Mucors, Alternarias, and yeasts. It was found that, when a fungus was 
sharply inhibited on normal cornmeal agar, it was better able to contend 
with its antagonistic neighbor on a medium rich in dextrose. On the other 
hand, in washed agar the inhibition became even more marked. On media 
‘poor in nutrients, the line of demarcation as between two closely related 
Helminthosporiums became much more distinct and had the same charac- 
teristics that it would have on cornmeal agar between two widely divergent 
forms. 

The longer a colony of any organism grows by itself on the surface of a 
nutrient medium, the larger and more vigorous it becomes. Such a large 
colony is just as effectually checked by the proximity of another organism 
as if the two contending forms were of equal age and vigor. Helmintho- 
sporium with a 120-hour start is inhibited by organisms nos. 31, 33, 34, and 
45, and the inhibition is of the same type as if they had been planted simul- 
taneously. 


TEXT Fic. 4. A, distortion of an Helminthosporium filament caused by the presence 
of organism no. 45. 3B, distortion of an Helminthosporium filament caused by Bacillus 
ramosus. C,a normal filament of Helminthosporium. 


Change in the mass of inoculum has much the same effect as difference 
in the time of inoculation. If the mass of inoculum is small, the inhibition 


177 AMERICAN JOURNAL OF BOTANY [Vol. 11, 


may not be as great and the reaction may be slower. There are exceptions 
to this statement. The smallest amount of inoculum possible, using or- 
ganisms nos. 31, 33, 34, and 45, produces the same effect as when a mass 
many times larger is used. 


INHIBITION CHARACTERISTIC OF VARIOUS GROUPS 


The great groups of fungi were studied critically to ascertain whether 
any of the types of inhibition previously described were peculiar to them. 

Thirty-five different species representative of the Schizomycetes were 
studied. The Schizomycetes are quite variable regarding the nature 
of their inhibitions. Some of the most powerful inhibitors belong to this 
class. These cause marked morphological disturbances and may do so 
even over considerable distances. On the other hand, most of the bacteria 
studied were quite inert, causing no inhibition, and being covered eventually 
by the hyphae of even slow-growing filamentous fungi. It appears from 
my experiments that the spore-formers as a rule are strong inhibitors. 
Actinomyces likewise exhibited strong inhibitory action against most fila- 
mentous fung?. 

The Phycomycetes have a tendency to grow superficially and to spread 
rapidly over the surface, burying all other fungous colonies beneath their 
advancing mycelium. Asa rule, they neither cause inhibition, nor are they 
inhibited. 

The Ascomycetes vary somewhat in the nature of their behavior toward 
each other and in the presence of other organisms. Asa rule their inhibitory 
powers are not great, though some of the yeasts rank high as inhibitors in 
this class. With respect to these powers no distinction could be made 
between the conidial and ascigerous forms of the same species. 

Few cultures of Basidiomycetes were used because of the difficulty in 
getting them to grow well in artificial media. Sporidial cultures of two. 
varieties of Ustilago violacea were found to be absolutely inert with respect 
to their inhibitory powers. Moreover, they were but little affected in the 
presence of other fungous forms, but were invariably overgrown by them. 

The Fungi Imperfecti, like the Ascomycetes, are quite variable respect- 
ing their inhibitory powers. While these powers never appear to be great, 
yet they are possessed by nearly all of the group to some degree. 

Text figure 5 illustrates the nature of the contacts when the various 
fungi were grown with each other. With but few exceptions, the closer the 
degree of relationship the less marked is the inhibition between the colonies. 
If two colonies of Helminthosporium grown from spores obtained from a 
single pure culture are permitted to grow in the same petri dish, they will 
eventually grow together with their fibers intermingling, giving only slight 
evidence of separate contact borders. Two colonies of the same species of 
Mucor, if they are of the same race, react similarly, as do also two colonies 
of Alternaria, of Acrostalagmus, of Botrytis, or of Fusarium. If the or- 


Mar., 1924] PORTER — CHARACTERS OF CERTAIN FUNGI 178 


asGth 


- 
=— 

< ye 
. 


a 


fe [hg apaat carn ls > 
Ce RADAR 
<-> 


TEXT Fic. 5. Reactions of fungi growing together on'cornmeal agar. The figures 
refer to numbers in the accession list. The arrows point to the direction of the reaction. 
The symbols are explained as follows: a, Antagonistic and producing distortion. 5, 
Slightly antagonistic. ‘c, Antagonistic at first only. d, Complete inhibition without dis- 
tortion. e, Spore-formation increased. Growth checked. No distortion. jf, Overruns. 
Arrow points to the colony overrun. g, Noinhibition. #,Growsaround. Arrow points to 
colony grown around. 7, Mycelium more abundant and raised at contact. 


ganisms are of distinct species, they usually betray that fact by some more 
or less distinct hiatus at contact such as distorted hyphae, color lines, more 
abundant sporulation, or a wider neutral zone through which the hyphae 
of the two colonies are unable to pass. 


TExT Fic. 6. A, parent colony of Alternaria. A’, sub-colony of A. B, bacterial 
form inhibiting A. No inhibition between A. and A’. 


179 AMERICAN JOURNAL OF BOTANY [Vol. 11, 


When the relationship is more distinct than that of species, the line of 
demarcation is usually still more marked. Text figure 6 illustrates this 
point. In this figure, A represents a parent colony of Alternaria. A’ isa 
sub-colony of A, and they grow together, merging so perfectly that it is 
impossible to tell where one colony leaves off and the other begins. On the 
other hand, the line between A and B, a bacterial form, is made clearly evi- 
dent by the increased sporulation at contact. The rule cannot be applied 
further than this, since one can not say that members of two orders are more 
antagonistic than are the members of two families. After reaching certain 
limits, degrees of antagonism are not distinct. , 


4 
TExT Fic. 7. In every case A represents organism no. 25; B represents organism no. 
26. Inz, C = organism 31; in 2, C = 8; in 3, C = 32; in 4, C = 12; and in 5, C = 10. 


Mar., 1924] PORTER — CHARACTERS OF CERTAIN FUNGI 180 


Two different fungi may not respond alike in the presence of other forms. 
This may be of diagnostic aid even when the fungi concerned are as closely 
related as varieties of the same species. Two varieties of Helminthosporium 
teres, nos. 25 and 26 of the accession list, illustrate this possibility. The 
colonies of these Helminthosporiums differ somewhat in appearance, zona- 
tion being more marked in the latter than in the former. The organisms 
were both inhibited by a number of organisms with which they were grown, 
but reacted differently with most of them. The difference was so great that 
one had no difficulty in distinguishing between the two varieties, judging 
solely from the reactions due to antagonism. Text figures 7 and 8 illustrate 
the differences described. When organisms nos. 25 and 26 were grown in the 
presence of organism no. 31, a bacterial colony, no. 25 is not inhibited 
while inhibition in no. 26 is characterized by cessation of growth and greater 
spore-formation. In the presence of organism no. 8, a yeast, no. 25 is in- 
hibited somewhat, as may be seen by the fact that toward the yeast colony 


TExT Fic. 8. In every case A represents organism no. 25; B represents organism no. 
26. Inz, C =-5;in 2, C = 29; in3 and 4, C = 33. 


181 AMERICAN JOURNAL OF BOTANY [Vol. 11, 


no. 25 presents a straight rather than a rounded outline. No. 26 is inhibited 
more sharply and with spore-formation increased. In the presence of 
organism no. 32, a bacterial form, much the same relations exist as with the 
case of the yeast just described. These relations are also much the same 
when the two Helminthosporiums are grown in the presence of organism 
no. 12, another bacterial form, or of organism no. 10, a yeast. Both or- 
ganisms nos. 25 and 26 are inhibited by organism no. 5, a Fusarium, although 
in this case the growth of no. 25 is more sharply checked than is the growth 
of no. 26. Little difference in inhibition is to be noted when the two are 
grown together in the presence of organism no. 29, an Alternaria. In the 
presence of organism no. 33, an arborescent-growing bacterium, no. 25 is 
sharply inhibited at a considerable distance with cessation of growth, dis- 
tortion of hyphae, and increased spore-formation toward the sides presented 
to the bacterial colony. On the other hand, no. 26 showed no evidence of 
inhibition in the presence of organism no. 33. Summarizing, no. 25 is in- 
hibited markedly only by organisms nos. 5, 29,and 33. No. 26 is markedly 
inhibited by organisms nos. 31, 8, 32, 12, 10,5, and 29. This experiment was 
repeated many times, invariably with the same results as described above 
and as shown by text figures 7 and 8. | 
Reference to the diagram (text fig. 5) reveals illustrations of the same 
sort. In this diagram attention is called particularly to reactions exhibited 


TExT Fic. 9, Pestalozzia inhibited by C, Penicillium colonies, but not by B, Penicil- 
lium colonies of a species different from C. 


by organisms nos. 36 and 37. In this instance Acrothecium caused dis- 
tortion, the threads being twisted into knots when this fungus was in contact 
with organism no. 36, an Helminthosporium; no. 37, another Helmintho- 
sporium, was slightly inhibited by the Acrothecium but without visible 
distortion. In this case no. 37 caused more abundant spore-formation in 
the Acrothecium colony. Actinomyces caused both Helminthosporiums 
to be inhibited, but distortion of filaments was produced only in no. 36... With 


Mar., 1924] © PORTER — CHARACTERS OF CERTAIN FUNGI 182 


organism no. 25, another Helminthosporium, growth was completely stopped 
in no. 36 with practically no intermingling of hyphae. Growth of no. 37 was 
slowed somewhat in the presence of organism no. 25, but the hyphae mingled 
to a considerable degree. 

Text figure 9 shows a culture of Pestalozzia. This became contaminated 
with two races or species of Penicillium. Three colonies of one kind of 
Penicillium caused no inhibition of the Pestalozzia. The other Penicillium, 
of which there were also three colonies, completely inhibited the Pesta- 
lozzia with increased sporulation and a piling up of the hyphae. These 
two kinds of Penicillium are clearly differentiated by the nature of their 
reaction with the Pestalozzia. 


BIOLOGICAL EQUILIBRIUM 


Inasmuch as organism no. 45 was found to possess such extreme in- 
hibitory powers, an effort was made to discover whether this organism might 
be of some practical importance in checking the growth of other fungi upon 
plants parasitized by them. 

In the first of this series of experiments, sterile wheat seedlings were 
grown in rag-dolls and the cloth around the seedlings was heavily inoculated 
with Helminthosporium spores. One half of the rag-dolls prepared in this 
manner had previously been immersed in a broth culture of organism no. 
45. Table 1 gives the results of this experiment. 


TABLE I. Protection afforded wheat seedlings from attacks by Helminthosporium, using 
organism no. 45 as the protecting agent 


October 25 November 3 December 2 December 18 
Per- Per- Per- Per- 
Infe N- | cent |Inf cent |Infec- _Un- cent |Infec- ,Un- cent 
d |imfec-linfec-| ted |™CImnfec-| ted [Mel Infec-| ted |'ME-| Infec 
ted ted ted ted 
Protected by no. 45....| I Se lOnta 4 GulaIOul Law a28" a sishig 1.30.) 13.04 
Unprotected << oo 0hare on 4 StA0; ees | SO 1L28 15 PGS. 1h 20 9 9 | 68.9 


Without either Helmin- 

thosporium or organ- 

hihiGpy Geeae gener On ereeAG? | sOn1 LO O Ox Pea Saiie C,0) 70 51525 9).'G:00 
Without Helmintho- 

sporium, with organ- 

ASTRO cei S rare area. oi! Oo | 10 oPApeta Gina de: 0) Get a0 tie 0.0) 6 Oe s0nL 0,00 


The percentages given in this table would indicate that a certain amount 
of protection was given to the wheat seedlings by the presence of organism 


no. 45. 


The next experiment was made to determine whether organism no. 45 
would protect plants undér more natural conditions than existed in the 
foregoing experiment. It was decided to attempt the protection of flax 
seedlings grown in pots from attacks of Fusarium lint. Fusarium lint grows 


183 AMERICAN JOURNAL OF BOTANY . [Volix} 


well in the soil, quickly infects and kills the plants, and in plate cultures 
was inhibited by organism no. 45, although not so strongly as are the Hel- 
minthosporiums. In this experiment the protected seedlings had their bases 
surrounded by cores of earth heavily impregnated with a broth culture of 
organism no. 45 and separated from the surrounding earth containing 
Fusarium lint by paper cones. The controls were surrounded by cores of 
sterile earth, also in paper cones. The earth in these cones was saturated 
with a sterile broth solution in order to have the physical conditions the 
same. All the plants in this experiment succumbed to Fusarium wilt. The 
controls, however, in every case showed indications of the wilt before the 
protected plants did. An examination of the protected plants indicated 
that the roots had penetrated the paper cones and were growing into the 
unprotected regions containing Fusarium. This fact probably accounts 
for the absence of protection exhibited by the inhibiting organism in this 
case. Further experimentation on this phase has been inconclusive because 
of difficulty in securing virulent strains of Fusarium lint. 

Experiments were made to determine whether organism no. 45 was as 
strongly inhibitory to Fusarium lini in soil as on culture plates. Soil thor- 
oughly moistened with beef bouillon was packed into test tubes. The 
lower third of the tube was inoculated with Fusarium lini, the middle 
third was moistened with a broth culture of organism no. 45, and the top 
third was sterile earth. In the controls, the middle third of the tube was 
filled with sterile earth. Samples were removed from time to time from 
the top third of each tube and plated. After a month, Fusartum lini had 
not penetrated to the top third of earth in any tube where the intervening 
third was impregnated with a suspension of organism no. 45. In most of 
the controls, after an interval of two to three weeks Fusarium lint could 
be detected by cultural methods in the upper third of earth. 

Plate cultures of earth were made, the earth being moistened with beef 
bouillon and then packed while damp into plates to the depth of a half 
centimeter or more. Transfers were made from pure cultures of fungi to 
the surface of the earth on these plates in the same manner as one would 
inoculate plates of agar. Most fungi grew nearly as well, although perhaps 
more scantily, on such plates as on plates containing cornmeal agar. Fusa- 
rium lint was as sharply inhibited by organism no. 45 on such a plate as on 
ordinary media. In fact, all fungous combinations tried reacted in much the 
same way as illustrated in text figure 5. On plain earth moistened with 
sterile water, most fungi grew so scantily that their inhibitions could only 
with difficulty be determined. On non-enriched earth, organism no. ‘45 
grows so slowly that it is doubtful whether it would be very useful in check- 
ing the normal soil flora. 

Potter’s (29) suggestion that plants may be protected from fungous 
attacks by injections of the metabolic by-products of the fungus concerned 
was thought worthy of testing at this time because it supports the idea that 


wi 


Mar., 1924] PORTER — CHARACTERS OF CERTAIN FUNGI 184 


inhibition is due to products secreted or excreted by the fungus rather than 
to the exhaustion of nutrients. Oranges were injected with the by-products 
of the growth of Penicillium and were then inoculated with Penicillium. 
Growth of the Penicillium was sharply inhibited at the line of injection and 
but slowly and imperfectly worked its way over the line. Oranges that had 
. not been injected with the staling solution were quickly and entirely covered 
with the Penicillium. 

Since there exists within the soil such a very close association between 
rootlets and fungi, the possibility exists that one may be very much affected 
or even considerably modified by the other. In order to determine how far 
this might be true, aseptic seedlings of barley, oats, wheat, and rye were 
placed in close proximity to colonies of Helminthosporium and of bacteria 
growing in petri dishes upon cornmeal agar. The seedlings were placed in 
such a manner that the tips of the rootlets of one and the sides of the root- 
lets of the other were presented to the fungous colony. The following 
possibilities were kept in mind: (1) Inhibition or stimulation of the fungus; 
(2) Inhibition or stimulation of root-hair production; (3) change of direc- 
tion of growth of the rootlet tip. After exposure for 48 hours there were 
no noticeable effects either upon the fungus or upon the rootlets (Plate VI, 
fig). | 


DISCUSSION 


Smith (40) summarizes the possibilities existent when two or more 
organisms are grown in close proximity as antagonistic, indifferent, or 
favorable. Zeller and Schmitz (48) enumerate the possibilities as stimulat- 
ing, inhibiting, overgrowing, and non-influencing. 

Garré (15), de Freudenreich (10), Laws and Andrews (21), Remy (37), 
Horrocks (19), and Frost (13), all working with Bacillus typhosus, have 
demonstrated that it may be inhibited in the presence of several other 
bacterial organisms. 

The antagonism of protozoa toward bacterial forms has been put to 
practical use in the purification of water and sewage. In this instance, 
however, the inhibitory action may be due to the actual ingestion of the 
bacteria by the other organisms involved. Purdy and Butterfield (32), 
Razetto (35), Huntemuller (20), Stokvis (45, 46), and Olitsky (27) have 
cited such examples. 

It has frequently been demonstrated during my experiments that some 
of the most profound inhibitory effects are exhibited by bacteria toward 
fungi. This fact has been noted by Ravn (33, 34), Elliot (12), and Rein- 
hardt (36). 

The antagonism which certain fungi exhibit against other fungi of the 
same or of different species has been noted in the literature by Blakeslee 
(3), Edgerton (11), Stevens and Hall (42, 43), Crabill (8), Reinhardt (36), 
Fulton (14), Zeller and Schmitz (48), F. L. Stevens (41), N. E. Stevens (44), 


185 AMERICAN JOURNAL OF BOTANY [Vol. 11, 


and among the higher fungi the same phenomenon is noted by Shantz and 
Piemeisel (38). ; 

The references to well authenticated instances in which fungi have 
stimulated growth of some sort or have been otherwise beneficial to each 
other are not so numerous. Nevertheless, such references are made by F. 
L. Stevens (41), Stevens and Hall (43), Shear (39), Zeller and Schmitz (48), 
Reinhardt (36), Ward (47), de Bary and Woronin (9), Manns (24), Prings- 
heim (31), and Nikitinsky (25). 

These effects, both antagonistic and stimulative, have a very direct 
bearing upon the members of a mixed culture and the nature of succession 
in such a culture. Pringsheim and Nikitinsky have expressed such views 
in articles already cited (31, 25). This idea is also upheld by Gwynne- 
Vaughan (16), Hesler (18), Smith (40), and Heinemann (17). 

The explanation of effects produced upon fungi in mixed cultures may 
be divided into two classes: (1) The nutrients of the medium may have 
become exhausted; (2) Products are formed which are detrimental or bene- 
ficial to further growth. Obviously when the nutrients are exhausted growth 
will be checked, but Leisegang (22) is one of the few who would explain the 
entire inhibitional phenomenon in this manner. On the other hand, many 
advocate the theory that during growth organisms give off materials that 
may be inhibitory or stimulatory to themselves or to other members of the 
flora. Gwynne-Vaughan (16), Clark (7), Brown (4), Balls (1), Fulton (14), 
Lutz (23), and Chambers (6) are advocates of the latter theory. 

By taking advantage of the products produced as mentioned in the 
preceding paragraph, fungous growth may be inhibited or stimulated to 
the advantage of man. Those who have done this experimentaily are Potter 
(29, 30), Picardo (28), and Beauverie (2). Norton (26) believes that such 
experiments have no practical value. 

Judging from my own experiments and from the literature on this 
subject, it would seem possible that under certain circumstances the knowl- 
edge of the relationships of fungi could be used in controlling the growth of 
these organisms to our advantage. 


SUMMARY 


1. The inhibitions exhibited by fungi mav be grouped into five classes. 

2. Helminthosporium was inhibited by various chemicals in a manner 
similar to that caused by other fungi. 

3. The inhibiting qualities of a fungus may be of aid in identification of 
species. 7 

4. The richer the medium in nutrients the less marked were the inhibi- 
tions. 

5. The inhibitions varied but slightly with changes in the amount of 
inoculum, in time of inoculation, or in depth of medium. 

6. A common cause of the inhibitory action in the cases studied was 
determined to be the presence of some product formed during growth. 


Mar., 1924] _ PORTER — CHARACTERS OF CERTAIN FUNGI 186 


7. Seedlings were protected measurably from infection by Helmintho- 


sporium, using organism no. 45. 


8. Flax seedlings were measurably protected from Fusarium, which 


could only with difficulty pass a layer of earth heavily infected with the 
inhibitor. 


9. Roots of seedlings and root hairs gave no tropic response in the pres- 


ence of fungi. 


PP ye 


23; 


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105-129. 1923. 


. Chambers, W. H. Bacterial inhibition. Germicidal action in milk. Jour. Bact. 


5: 527-541. 1920. | 
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. Clark, J. F. On the toxic properties of some copper compounds with special reference 


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AMERICAN JOURNAL OF BOTANY. VOLUME XI], PLATE IV. 


PORTER: CHARACTERS OF CERTAIN FUNGI 


AMERICAN JOURNAL OF BOTANY. VOLUME XI, PLATE V. 


PORTER: CHARACTERS OF CERTAIN FUNGI 


AMERICAN JOURNAL OF BOTANY VOLUME XI, PLATE VI. 


PORTER: CHARACTERS OF CERTAIN FUNGI 


Mar., 1924] PORTER — CHARACTERS OF CERTAIN FUNGI 188 


EXPLANATION OF PLATES 


PLATE IV 


Fic. 1. Alternaria inhibited by two colonies of organism no. 45. 
Fic. 2. Helminthosporium in the center inhibited on three sides by Actinomyces. 
Fic. 3. Helminthosporium in center inhibited by four small colonies of organism no. 
45. 
EEATE RV 


Fic. 1. Nature of reaction when two colonies of the same species grow together. 
Organism no. 25 of the accession list. 

Fic. 2. Nature of the reaction when two species of Helminthosporium grow together. 
Organisms nos. 25 and 37 of the accession list. 


PLATE VI 


Fic. 1. Helminthosporium inhibited by a block of sterile agar taken from the vicinity 
of organism no. 45. 


Fic. 2. Indifference of wheat rootlets to a colony of Helminthosporium. 


VACANT 
3 2662874 


0112 07 


VITA 


The writer of this thesis was born at Mackinaw, Illinois, February 22, 
1889. His early education, including his high school training, was re- 
ceived in the schools of that community. In 1907 he entered the Illinois 
Wesleyan University at Bloomington, receiving the degree of B.S. from 
that institution in 1911. In the spring of 1912 he entered the University 
of Illinois, graduating in 1913 with the degree of A.B., doing most of his 
work in the College of Agriculture. From 1914 to 1916 he was instructor 
in biology at Parsons College, Fairfield, lowa. From 1916 to 1919 he 
served as head of the Department of Biology at Fairmount College, 
Wichita, Kansas. He served this college as registrar in 1918. The sum- 
mer and latter part of 1918 was spent in military service. In 1919 he again 
entered the University of Illinois to do graduate work in the Department 
of Botany. He was assistant in botany at the University of Illinois 1919- 
1921, including the summer terms of I920 and I921. He received the de- 
gree of Master of Arts in 1921. From September, 1921 to September, 1922 
he was engaged part time as botanist in the Natural History Survey and 
part time in graduate work. The summer of 1922 was spent in the field in 
the interests of the Natural History Plant Disease Survey. The university . 
year of 1922-1923 was devoted to full-time graduate work. 


